1. References (All Incorporated by Reference)
The following can be referred to as background in order to aid in understanding of certain of the terms and expressions below.                U.S. Patent Application (BioArray Solutions, hereinafter sometimes referred to as “eMAP®”): “Multiplexed Analysis of Polymorphic Loci by Concurrent Interrogation and Enzyme-Mediated Detection” filed Oct. 15, 2002; Ser. No. 10/271,602;        U.S. Patent Application (BioArray Solutions): “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays” filed on Aug. 23, 2002, Ser. No. 10/204,799 (discussing Random Encoded Array Detection, READ™);        U.S. patent application (BioArray Solutions): “Multianalyte Molecular Analysis Using Application Specific Random Particle Arrays” filed on Dec. 28, 2001; Ser. No. 10/032,657        U.S. Patent Application (BioArray Solutions, hereinafter sometimes referred to as “Multiplexed Expression Profiling”): “Optimization of Gene Expression Analysis using Immobilized Capture Probes,” filed Oct. 26, 2004, Ser. No. 10/974,036, (including discussion therein relating to subtractive differential gene expression analysis; in the disclosure in the present application, sense and anti-sense strands are produced by incorporation of RNA pol promoter sequences);        U.S. Patent Application (BioArray Solutions): “Arrays of Microparticles and Methods of Preparation Thereof,” filed on Jul. 9, 2002; Ser. No. 10/192,352        U.S. Patent Application (BioArray Solutions hereinafter sometimes referred to as “PARSE™”): “System and Method for Programmable Illumination Pattern Generation,” filed Jan. 24, 2001, Ser. No. 09/768,414        U.S. Pat. No. 6,251,691 (BioArray Solutions, hereinafter sometimes referred to as “LEAPS”): “Light Controlled Electrokinetic Assembly of Particles Near Surfaces”: see especially FIG. 8;        U.S. Patent Application (BioArray Solutions hereinafter sometimes referred to as “Solvent Tuning”): “Method for controlling solute loading of polymer microparticles” filed Jan. 21, 2003, Ser. No. 10/348,165        U.S. Pat. No. 5,759,820 (Dynal AS) “Process for Producing cDNA”        European Patent No. 0 368 906 B2 (Gingueras et al.; discussing isothermal, exponential amplification (“3SR”))        Guatelli et al, “Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral amplification”, Proc. Nat'l Acad. Sci. USA 87, 1874-1878 (March 1990) (discusses invoking RNAseH activity)        Kwoh et al, “Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format”, Proc. Nat'l Acad. Sci. USA 86, 1173-1177 (February 1989) (discussing transcription-mediated amplification using thermal cycling)        U.S. Pat. No. 5,399,491 (Kacian et al.; discussing isothermal, exponential amplification) T Kievits et al., J Virological Meth 35 (Issue 3), December 1991, pp 273-286; EP 273086 (discussing NASBA)        U.S. Pat. Nos. 5,716,785 and 5,891,636 (Van Gelder et al.); Van Gelder et al, “Amplified RNA synthesized from limited quantities of heterogeneous cDNA”, PNAS 87, 1663-1667 (March 1990);        Krieg & Melton, Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs, Nucleic Acids Res 12, 7057-707 (1984) (discussing use of SP6, T4, T7 promoter sequences)        Fermentas Life Sciences Website and references listed there (referred to in Ex. 1) include:        Melton, D. A., et al., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nucleic Acids Res.,12, 7035-7056, 1984.        Church, G. M., Gilbert, W., Genomic sequencing, Proc. Natl. Acad. Sci. USA, 81, 1991-1995, 1984.        Peebles, C. L., et al., A self-splicing RNA excises an intron lariat, Cell, 44, 213-223, 1986.        Melton, D. A., Injected antisense RNAs specifically block messenger RNA translation in vivo, Proc. Natl. Acad. Sci. USA, 82, 144-148, 1985.        Krainer, A. R., et al., Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro, Cell, 36, 993-1005, 1984.        Witherell, G. W., et al., Cooperative binding of R17 coat protein to RNA, Biochemistry, 29, 11051-11057, 1990.        Bernstein, E., et al., Role for bidentate ribonuclease in the initiation step of RNA interference, Nature, 409, 363-366, 2001.        U.S. Pat. No. 6,013,431 (Soderlund, Syvanen)        Pastinen T., et al., A system for specific, high-throughput genotyping by allele-specific primer exrtension on micorarrays. Genome Research 2000 10:1031-1042        
2. Discussion
Existing protocols for multiplexed interrogation of nucleic acid configurations—for example, those discussed in patent application Ser. No. 10/032,657 which use the Random Encoded Array Detection (READ™) format, where a bead array is formed on a substrate (a “BeadChip™”)—generally invoke the conventional sequence of assay steps, i.e., separate DNA extraction and capture, amplification, post-amplification “clean-up” and finally, analysis by hybridization-mediated detection or capture-mediated probe elongation. In the READ format, assay signals reflecting the interaction of target(s) with an array of bead-displayed probes are generated on-chip and are recorded, by “snapshot” imaging of the array, without intervening sample transfers. This combination of analysis and read-out simplifies the protocol, decreases the time to completion of the assay and increases sample throughput.
The READ™ format is well-suited for the realization of miniaturized assays permitting rapid multiplexed analysis because an array of 10,000 microparticles (or “beads”), each of 3 micron diameter, occupies an approximate volume of only 300×300×5 μm3 or ˜0.5 nanoliters. Reducing assay volumes to nanoliter scale provides the advantage of requiring only small amounts of reagents generally, and requiring only a small number of target molecules for analysis. However, a significant set of problems arise on such miniaturization, notably including the design of functionally integrated assay protocols that combine multiple reaction steps in a single reaction without requiring intervening steps of sample manipulation.
Changing assay temperature, if needed to accommodate PCR for example, can cause significant evaporation and a correspondingly negative effect on the assay conditions. Methods of isothermal amplification, including the Transcription Amplification System (“TAS”) described by Gingeras et al. and others (see references above) have the advantage of largely eliminating design constraints arising from the variation in effective “melting”-temperatures of the amplification products (see U.S. Ser. No. 10/974,036, Multiplexed Expression Profiling, for discussion regarding effective melting temperatures). Isothermal amplification also eliminates the need for carefully controlled heating and rapid cooling of the reaction in each cycle to avoid excessive evaporation and unacceptable increases in solute concentration in the reaction mixture. The elimination of the rapid cooling step eliminates a cumbersome constraint from the design of the reaction vessel for integrated reaction protocols, which otherwise must provide for rapid heat transfer to an active cooling device while permitting optical access to the reaction, so as to permit recording or real-time monitoring of the reaction products and assay results. However, for TAS and related methods of exponential isothermal amplification, performance critically depends on maintaining strict assay conditions including (constant) temperature, pH and ionic strength, including allowing only a limited range of probe and primer lengths. When contemplating homogeneous multiplexed formats of TAS, capture probess and primers for reverse transcription and second strand cDNA synthesis both bind to RNA generated in the IVT step, and this can reduce the amount of the anti-sense RNA product detected. These limitations make the TAS difficult to multiplex, as is needed in commercial applications.
With linear IVT amplification, performed in the conventional formats which typically call for tens of microliters of analyte solution, it is questionable if, in a reasonable time-frame, a sufficient amount of product is generated to obtain a detectable assay signal. One would generally have a sample of genomic DNA sample for the IVT assay, in low concentration, meaning that in a small volume, there would be relatively few molecules in total. Thus, a miniaturized format presents a special set of problems, which must be solved to have an effective process for use in a commercial setting.
Other problems with miniaturization of an IVT or TAS system relate to the mixing of the assay reagents, which cannot be accomplished with conventional mechanical methods due to the small volume involved. Diffusion alone must be relied upon to provide sufficient mixing in a sufficiently short period of time to bring assay reagents into contact such that they can react and form a detectable amount of product in the available time for completion of the assay.
An additional challenge arises because in a miniaturized format, the assay system needs to be “closed” to minimize contamination and evaporation. Thus, for a miniaturized assay, as it is difficult to monitor the reaction and correctly time subsequent addition of reagents throughout reaction, and with conventional apparatus, it is impractical to add nano-liter quantities of reagents in the course of the reaction. This can be accomplished with an integrated homogeneous assay format conducted in a hermetically sealed reactor, in which reagents are not added during the reaction process, but rather are all placed in the reaction solution at the start of the reaction process.
Assay integration in hermetically sealed reactors is desirable to minimize the number of manipulations, hence risk of operator error, and to minimize assay contamination risk, and the risk of contamination of the assay operator (which is especially significant in viral load assays).